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The roles of pDC and IFN-α have not been well defined in IgA nephropathy (IgAN). In this study, we investigated the abundance of pDCs and IFN-α in IgAN patients and the response of peripheral blood mononuclear cells (PBMCs) after stimulation of the pDC-preferred TLR9 ligand CpG2216. The effects of IFN-α on plasma cell differentiation and leukocyte migration were also investigated. Here, we found that the percentages of pDCs were increased in PBMCs of IgAN patients, than in those of healthy controls. Plasma levels of IFN-α proteins and abundance of plasma cells were higher in IgAN patients than in healthy donors. Plasma IFN-α levels were positively associated with proteinuria, renal IgM deposition, and renal tubular atrophy/interstitial fibrosis grade in IgAN patients. Ex vivo activation of TLR9 on pDCs resulted in increased IFN-α production and enhanced plasma cell differentiation in IgAN patients as compared with healthy donors. IFN-α treatment led to increased plasma cell differentiation in vitro. IFN-α also significantly promoted expression of chemokines IP-10 and MCP-1 in human mesangial cells, which subsequently facilitated the transendothelial migration of human CD4+ and CD14+ cells. In conclusion, pDC and its secreted cytokine IFN-α may play important roles in pathological changes of IgA nephropathy. [ABSTRACT FROM AUTHOR]

Abstract: Vaccines produced in plant systems are safe and economical; however, the extensive application of plant-based vaccines is mainly hindered by low expression levels of heterologous proteins in plant systems. Here, we demonstrated that the post-transcriptional gene silencing suppressor p19 protein from tomato bushy stunt virus substantially enhanced the transient expression of recombinant SARS-CoV nucleocapsid (rN) protein in Nicotiana benthamiana. The rN protein in the agrobacteria-infiltrated plant leaf accumulated up to a concentration of 79μg per g fresh leaf weight at 3 days post infiltration. BALB/c mice were intraperitoneally vaccinated with pre-treated plant extract emulsified in Freund''s adjuvant. The rN protein-specific IgG in the mouse sera attained a titer about 1:1,800 following three doses of immunization, which suggested effective B-cell maturation and differentiation in mice. Antibodies of the subclasses IgG1 and IgG2a were abundantly present in the mouse sera. During vaccination of rN protein, the expression of IFN-γ and IL-10 was evidently up-regulated in splenocytes at different time points, while the expression of IL-2 and IL-4 was not. Up to now, this is the first study that plant-expressed recombinant SARS-CoV N protein can induce strong humoral and cellular responses in mice. [Copyright &y& Elsevier]

Abstract: Site-specific proteases are the most popular kind of enzymes for removing the fusion tags from fused target proteins. Nuclear inclusion protein a (NIa) proteases obtained from the family Potyviridae have become promising due to their high activities and stringencies of sequences recognition. NIa proteases from tobacco etch virus (TEV) and tomato vein mottling virus (TVMV) have been shown to process recombinant proteins successfully in vitro. In this report, recombinant PPV (plum pox virus) NIa protease was employed to process fusion proteins with artificial cleavage site in vitro. Characteristics such as catalytic ability and affecting factors (salt, temperature, protease inhibitors, detergents, and denaturing reagents) were investigated. Recombinant PPV NIa protease expressed and purified from Escherichia coli demonstrated efficient and specific processing of recombinant GFP and SARS-CoV nucleocapsid protein, with site F (N V V V H Q▾A) for PPV NIa protease artificially inserted between the fusion tags and the target proteins. Its catalytic capability is similar to those of TVMV and TEV NIa protease. Recombinant PPV NIa protease reached its maximal proteolytic activity at approximately 30°C. Salt concentration and only one of the tested protease inhibitors had minor influences on the proteolytic activity of PPV NIa protease. Recombinant PPV NIa protease was resistant to self-lysis for at least five days. [Copyright &y& Elsevier]

Na~/H~ antiporters (NHX) are ubiquitous transmembrane proteins that play a key role in salt tolerance of plants. In this study, the sequence of 3 Arabidopsis NHX genes (AtNHX2-4) were compared with other AtNHX members. Putative cis-elements analysis identified elements that have been associated with stress responses. The activities of the promoters AtNHX2-4 were studied in transgenic plants carrying corresponding promoter-~-glucuronidase (GUS) fusions. The AtNHX2 promoter-GUS analysis indicated that AtNHX2 was expressed in constitutive pattern with high GUS activity in roots and leaves. AtNHX2 promoter activity was not up-regulated by NaCI or abscisic acid (ABA), in contrast to the AtNHX1 promoter which was previously studied. The AtNHX3 and AtNHX4 promoters showed tissue-specific activities. Strong GUS activity was detected in roots and vascular bundles of the stele in plants carrying an AtNHX4 promoter-GUS fusion, and GUS activity increased under salt stress suggesting a function related to salt tolerance. Transgenic plants carrying the AtNHX3 promoter-GUS fusion showed strong GUS activity in petals, stamens and tops of siliques, suggesting a possible role of AtNHX3 in flower and seed development. Results of histochemical analysis suggested that AtNHX2-4 are involved in divergent functions and are differentially regulated under abiotic stress. The structure of AtNHX4 was predicted to include 12 transmembrane regions and a NHX domain. Overexpression of AtNHX4 in Arabidopsis transgenic lines confers greater salt tolerance than in wild type plants. These results suggest that AtNHX4 may encode a putative vacuolar NHX that plays an important role in salt tolerance. [ABSTRACT FROM AUTHOR]

Background: B cell activating factor belonging to the TNF family (BAFF) is vital for B cell survival, proliferation and activation. Evidence indicates that BAFF is systemically or locally increased in glomerulonephritis (e.g. lupus nephritis, IgA nephropathy). However, the effect of BAFF on human mesangial cells is not known. Methods: The impact of BAFF on the proliferation of a human mesangial cell line in vitro was investigated. The expression of BAFF receptor (BAFF-R) and downstream signal transduction were explored. The influence of BAFF on the expression of related genes was also studied. Results: Our data indicated that BAFF had a proliferative effect on human mesangial cells, as supported by the results of cell proliferation assays and the inhibited expression of the pro-apoptotic gene Bim. BAFF-R was expressed on the cell membrane of human mesangial cells and blockade of BAFF/BAFF-R binding abrogated the proliferative effect of BAFF on human mesangial cells. BAFF stimulation led to rapid phosphorylation of NF-κBp65, Akt and MAPK p38 kinase in human mesangial cells, whereas it had no effect on the expression of NF-κB p100 and phosphorylation of Erk. The phosphorylation of Akt was very sensitive to blockade of BAFF/BAFF-R ligation, although activation of MAPK p38 and NF-κBp65 was not. BAFF treatment resulted in decreased expression of BAFF-R, which implied negative feedback regulation after its binding. Conclusions: BAFF promoted proliferation of human mesangial cells, which was mediated via BAFF-R. The BAFF/BAFF-R interaction triggered Akt, p65 and p38 activation, with Akt phosphorylation being tightly dependent on BAFF/BAFF-R interaction. [ABSTRACT FROM AUTHOR]

TLR7 has been linked to the pathogenesis of glomerulonephritis, but its precise roles are not clear. In this study, we evaluated the roles of TLR7 in IgA nephropathy (IgAN). TLR7 proteins were abundant in CD19+ B cells infiltrated in the kidneys of patients with IgAN. The intensities of both intrarenal TLR7 and CD19 proteins were closely associated with kidney function (estimated glomerular filtration rate [eGFR] and serum creatinine concentration) and renal histopathology (tubular atrophy, leukocyte infiltration, tubulointerstitial fibrosis, and global glomerulosclerosis) in patients with IgAN. Meanwhile, TLR7 mRNA levels were significantly increased in peripheral blood B cells of patients with IgAN. TLR7+CD19+ B cells expressed inflammatory cytokines (IL-6 and IL-12) in kidneys and produced high levels of IgA1 and galactose deficient-IgA1 (Gd-IgA1) in peripheral blood of patients with IgAN. Mechanistically, TLR7 activated B cells to produce high levels of Gd-IgA1 via the TLR7-GALNT2 axis in IgAN. Protein levels of GALNT2 were increased by overexpression of TLR7, while they were reduced by TLR7 knockdown in B cells. GALNT2 overexpression augmented Gd-IgA1 production in B cells derived from patients with IgAN. Taken together, high TLR7 expression in B cells has dual roles in the development and progression of IgAN, by facilitating renal inflammation and Gd-IgA1 antibody synthesis.

B cells are critically important for the pathogenesis of IgA nephropathy (IgAN). The present study aimed to investigate the abundance of B cell activating factor (BAFF), which belongs to the tumor necrosis factor superfamily, in the peripheral blood of patients with IgAN. The different forms of BAFF in peripheral blood and its association with clinical features and immunological factors were analyzed. mRNA levels of BAFF and other associated genes in the peripheral blood mononuclear cells (PBMCs) of patients with IgAN and controls were analyzed by quantitative polymerase chain reaction. Cellular BAFF proteins in PBMCs and plasma soluble BAFF proteins were measured by western blot analysis and ELISA, respectively. PBMCs from patients were stimulated with Streptococcus pyogenes (S. pyogenes) ex vivo for the BAFF secretion assay. The data demonstrated that, although mRNA levels of BAFF in PBMC were not significantly increased in patients with IgAN, they were positively associated with those of a proliferation inducing ligand (APRIL), Toll‑like receptor (TLR)2, TLR4 and TLR7. The cellular BAFF protein in PBMCs was not upregulated. Plasma BAFF protein levels in patients with IgAN (n=76) were significantly decreased compared with controls. However, plasma BAFF levels were positively associated with serum creatinine, proteinuria, uric acid and group A Streptococcus infection index in patients with IgAN. In patients with IgAN, plasma BAFF concentrations were markedly higher in those with more severe renal tubular atrophy/interstitial fibrosis and global glomerulosclerosis. Furthermore, BAFF production in PBMCs of patients with IgAN was increased following S. pyogenes stimulation ex vivo. In conclusion, plasma BAFF levels in patients with IgAN were associated with renal function and disease activity. S. pyogenes infection was closely associated with BAFF production in patients with IgAN.

IRF7 is known as the master regulator in virus-triggered induction of type I IFNs (IFN-I). In this study, we identify GBP4 virus-induced protein interacting with IRF7 as a negative regulator for IFN-I response. Overexpression of GBP4 inhibits virus-triggered activation of IRF7-dependent signaling, but has no effect on NF-κB signaling, whereas the knockdown of GBP4 has opposite effects. Furthermore, the supernatant from Sendai virus-infected cells in which GBP4 have been silenced inhibits the replication of vesicular stomatitis virus more efficiently. Competitive coimmunoprecipitation experiments indicate that overexpression of GBP4 disrupts the interactions between TRAF6 and IRF7, resulting in impaired TRAF6-mediated IRF7 ubiquitination. Our results suggest that GBP4 is a negative regulator of virus-triggered IFN-I production, and it is identified as a novel protein targeting IRF7 and inhibiting its function.